Concentration of fat-soluble vitamins



Patented Nom i946 STAT CONCENTRATIONOFFAT-SOLUBLE VITAMINS Edgar M. Shantz, Rochester, N. Y., assignor to Distillation Products, Inc., Rochester, N. Y., a

corporation of Delaware No Drawing. Application January 21, 1944,

Serial No. 519,203

Claims.

This invention relates to improved procedure for the concentration of fat-soluble vitamins and in particular for the concentration of vitamin A contained in fish liver oils, and fractions thereof.

This invention has for its object to provide improved procedure for the concentration of fatsoluble vitamins. Another object is to provide improved procedure for the concentration of vitamins contained in. fish liver oils and distillates .orfractions thereof, and in particular for the concentration of vitamin A 'containedin such oils or fractions. Other objects will appear herethese are set forth for the purpose of illustration and not in limitation thereof.

The amine, or ammonium base, which is used must be one which will substantially completely react with the glyceride oil under the conditions of treatment. Primary ami es are preferred since they react with the glyceride oil with relative rapidity. Secondary amines react somewhat more slowly but can be used satisfactorily. Quaternary ammonium bases may be used but tertiary amines are unsatisfactory. Substituted amines, or ammonium bases, such as hydroxy, halide, etc., amines can be used and are to be understood asbeing included within the expressions primary amines, "secondary amines, and quaternary ammonium bases as used herein and in the claims.

The reaction takes place over a relatively wide range of temperature. The preferred range is about 80 to about 150 C. For most purposes a temperature of about 100 C. is quite satisfactory. Too high a temperature causes decomposition of the more labile vitamins whereas low temperatures decrease the rate of reaction.

The time of reaction depends upon factors such as concentration of the reactants, the temperature, and the particular ammonium derivative used. In most cases the reaction requires from about two to twenty hours.

At least one molecular equivalent of amine should be used based on the fatty acid content of the glyceride treated if complete removal of the fat is required. It is more practical and preferred to use a slight excess. To speed up the reaction, from two to ten molecular equivalents of amine may be used for a given amount of oil.

Excess reagent may, of course, be recovered and used again.- It is also preferred to carry out the reaction in an ,inert atmosphere such as carbon dioxide or nitrogen.

If a diamine is used, it can be made to react with two molecules of fatty acid to yield a .di-.

amide. One method of doing this is to use just enough of the diamine or polyamine to provide one NH2 group for each fatty acid radical present in the fat which contains the vitamin. Another method is to react a given quantity of fat with an excess of the diamine, after whichthe excess unused diamine is removed and the reaction product is then allowed to react with another equal quantity of the same oil or fat.

If it is desired, the reaction products may be treated with an esterifying agent suchas acetic ,anhydride or acetyl chloride before distillation, solvent extraction, or filtration of the desired "unsaponiflable fraction in order to formvitamin esters. Such a reagent would also react with certain of the fatty acid substituted amides, "as

will be shown below. This is desirable in certain cases to decrease their solubility.

The following empirical formulas are for the purpose of illustrating and clarifying the .reac-' Specific examples are tions described above. given later.

1. Reaction of an oil with a primary amine,

2. Reaction of oil with a hydroxy amine, such as ethanolamine:

R'COO Hz glycerol 3RICONHCHQCHEOH 3. Reaction of above products with esterifying agent such as acetyl chloride:

" R'COO 3 4. Reaction of oil with an excess of a polyamine such as ethylenediamine:

a coocni a NmcmcmNn,

5. Reaction of above products with another equal amount of oil:

3R'd0NHCHICHiNH: R'COOCHI R'COO a'coo In glycerol SR'CO'NHCHSCHSNHCOR' 8. Reaction of products of illustration 4 with an esterifying agent such asacetyl chloride:

onion non 3R'CONHCH:CH2NH| acmooci -i GHQOCOCHI HOCOCH; 3R'CONHOH2CH2NHCOCHI 6H0] HrOCOCH;

7. Reaction of two equivalents of oil with a polyamine such as ethylenediamine:

n'coocm 2 R'COO H aNmcmcmNm R'cooom 8. Reaction of an oil with a quaternary ammonium base:

a'coocrn R'c'oocir auz'mNon -o glycerol an'ooomn'qi a'coo H:

2c. Reactionof vitamin esters with a hydroxy amine, such as ethanolamine:

3a, Reaction of above products with esterifying agent such as acetyl chloride: L ROI-1+R'CONHCHaCH2OH+2CI-isC0Cl 4a. Reaction of vitamin esters with an excess of a polyamine such as ethylenediamine:

v a'coo'n- -nmcrnonmm ROHi-i-R'CONHCHzCHzNHa 5a. Reaction of above products with another equal amount of oil:

R'CONHCI-IzCHzNHa-l-R'COOR ROH-l-R'CONHCI-hCI-IaNHCOR' 6a. Reaction of products of illustration 4 with an esterifying agent such as acetyl chloride:

glycerol aa'c ONHCHICHzNHt amine was removed under reduced pressure and the residual product distilled in a cyclic molecular J 4 7a. Reaction of two equivalents oi vitamin esters with a polyamine such as ethylenediamine:

The following are specific examples of the methods outlined above.

Example 1- 190 grams of fish liver oil distillate containing 210,000 units per gram of vitamin A ester were heated at C. under nitrogen for twenty hours with 700 cc. of ethanolamine. At the end of this time the excess ethanolamine was removed under reduced pressure. The product was a waxy solid at room temperature but became completely liquid at 50 to 60 C. 183 grams of this material were distilled in a cyclic molecular still. A fraction weighing 32.2 gms. was obtained which distilled between 100 and C. at a pressure of 3 to 10 m This fraction, a light yellow oil which became solid at room temperature, contained 900,000 units per gram of vitamin A alcohol.

Example 2 230 grams of fish liver oil distillate containing 200,000 units per gram of vitamin A ester were heated for 15 hours at 100 C. under nitrogen with 320 grams of ethanolamine. After removal of the excess ethanolamine under reduced pressure, the residual product was distilled in a cyclic molecular stilL The fraction distilling between 100 C. and C. at a pressure of 3 to 10a weighed 52 grams and contained 610,000 units of vitamin A alcohol per gram.

This distillate was then dissolved in 200 cc. of methyl alcohol and chilled to -60 C. to freeze out the fatty acid ethanolamides which had distilled with the vitamin A. After filtration in the cold, the methyl alcohol was evaporated and the residue was found to consist of 20 grams of a dark red oil containing 1,300,000 units of vitamin A per gram.

Example 3 grams of fish liver oil distillate containing 200,000 units of vitamin A per gram were heated for 16 hours at 100 C. under nitrogen with 450 cc. of ethanolamine. After removal of the excess ethanolamine by distillation under reduced pressure, the residue was taken up in 1.5 liters of warm ethyl ether. About 30% of the material was insoluble at room temperature and was filtered off. The filtrate was chilled to 5 C. and again filtered. The ether was removed, the residue dissolved in 500 cc. of methyl alcohoL'and chilled to 35 C. After filtration the methyl alcohol was evaporated and the residue was found to contain 54 grams of dark red oil having a potency of 508,000 units of vitamin A per gram.

Example 4 200 grams of fish liver oil distillate containing 200,000 units per gram or vitamin A were heated for 12 hours with 300 grams of 1-amino-2-butanol at 100 C. under nitrogen. The excess still. Four fractions weighing a total of grams were removed between 100 C. and C.

Example 5 200 grams of fish liver oil distillate containing 200,000 units per gram of vitamin A were heated.

- for 12 hours at 100 C. under nitrogenwith 275 grams of 2-amino-2-ethyl-L. 3-propanediol. The entire mixture was placed in the molecular still and the excess amine removed at 100 C. at

a pressure of about 1- mm. .The pressure was then reduced to 540;, and the temperature raised to 140 C. A distillate with a vitamin A potency of 400,000 units per gram was obtained. Example 6 275 grams of fish liver oil distillate were heated for'five hours at 100 C. with 2'75 grams of ethylene diamine. The excess ethylene diamine was removed under reduced pressure, and the residue distilled in the molecular still. ,The distillate had a potency of 363,000 units per gram of vitamin A.

Example 7 110 grams of fish liver oil distillate containing 200,000 units of vitamin A per gram were heated under nitrogen for hours at 100 C. with 100 grams of ethylene diamine. The excess diamine was removed under reduced pressure and 110 grams more of the fish liver oil were added to the residue. This mixture was heated for another twenty hours under nitrogen after which it was distilled in a molecular still. The product remained liquid only if heated to about 100 C. A distillate was obtained between 100 and 160 C. which was a viscous orange oil containing 570,000 units per gram of vitamin A.

Example 8 150 grams of fish liver oil distillate containing 200,000 units per gram of vitamin A were heated at 100 C. with 150 grams of ethylene diamine for hours. The excess diamine was removed under reduced pressure and the residue dissolved in one liter of acetone. 100 grams of pyridine, followed by 100 grams of acetic anhydride, were added. Upon cooling, the fatty acidacetic acid diamides of ethylene diamine crystallized out and. were filtered ofi. Ethyl ether was added to the filtrate and the solution washed several times with water. Upon evaporation of the ether, 68 grams of residue remained which contained 358,000 units per gram of vitamin A acetate.

Example 9 grams of fish liver oil distillate (200,000 units per gram), 30 cc. of tetraethanolammonium hydroxide aqueous solution) and 30 cc. of ethyl alcohol were refluxed for 2 hours under nitrogen. The mixture was dissolved in ether, washed three times with water to remove the reaction products which are soluble in water and the ether removed by evaporation; The residue consisted of 12 grams of red oil which contained 465,000 units per gram of vitamin A alcohol.

Example 10 270 gms. of fish liver oil distillate containing 186,000 units of vitamin A per gram were heated at C. for 15 hours with 160 gms. of aminoethylethanolamine under a stream of nitrogen. The reaction mixture was then placed in a molecular still and distilled. The first fraction, removed at C. at a pressure of 1 mm., contained mainly glycerol and excess aminoethylethanolamine. The fraction distilling between .4120 and 140 C. at a pressure of 15 microns consisted of a light yellow olid having a vitamin A potency of 5105000 units per gram.

Example 11 560 gms. of soupfin shark liver oil distillate having a vitamin A potency of 213,000 units per gram were heated withgms. of ethanolamine for 16 hours at 80 C under. nitrogen. The reaction mixture was then dissolved in 2.5 liters of. acetone and the fatty acid ethanolamides filtered off after successively chilling the mixture to 0 C., -'35 C., and 50 C. The solvent was then removed from the filtrate under reduced'pressure and the residue distilled in a.

molecular still. The fraction distilling between 110 and C. contained vitamin A alcohol a a potency of 620,000 units per gram.

Example 12 600 gms. of fish liver oil having a vitamin potency of 27,000 units per gram were heated under nitrogen for 16 hours at 80 C. with 100 gms. of ethanolamine. The reaction mixture was .dissolved in 3 liters of warm acetone. This solution was cooled to l5 C. and the solid material filtered ofi, washing with 500 ml. of chilled acetone. The filtrate was then chilled to -50 C. and again the solids which separatedwere filtered off as before. The solvent was removed from the filtrate by distillation and the residue distilled in a molecular still. The main fraction contained 416,000 units per gram of vitamin A.

Example -1 3 600 gms. of a high vacuum distillate of deodorizer scum containing 24% tocopherol were heated for 12 hours at 80 C. with 100 gills. of ethanolamine. 3.0 liters of acetone were added to the reaction mixture. The solution was then chilled to 50 C. and the separated solids filtered I oil". The solid material was dissolved in 2 liters of fresh acetone and again the solids were removed by filtration after chilling to -50 C. The filtrates were combined and the acetone removed by distillation. The residue was distilled in a molecular still. The fractiondistilling at C. contained 25% of the starting tocopherol at a 0 concentration of 50%. The fraction distilling at C. contained 45% of the original tocopherol at a concentration of 72.5%.

Example 14 izer scum containing 18% tocopherol were heated at 80 C. for 15 hours with 70 grams of ethanolamine. The reaction mixture was dissolved in 2 liters of acetone and the solid material removed 60 by filtration after chilling first to -20 C., then to -5o o. The acetone was distilled from the filtrate and the residue dissolved in 1 liter of ethyl ether. The ether solution was washed three times with lNHCl, followed by two water washes.

65 This washing was found to improve the taste of 400 gms. of a high vacuum distillate of deodoror pressing of the reaction products may be used.

Combinations-oi these separation procedures can be used.

While the procedure of my inventionis of particular advantage for the concentration or separation oi vitamins, it can also be used to separate or concentrate sterols as well as like materials which are present in fats and which do not react with the amines or like compounds dis closed herein.

What I claim is:

1. They process of preparing a fat-soluble vitamin concentrate which comprises reacting a glyceride fat which contains a fat-soluble vitamin with at least approximately stoichiometric amounts of a member of the group consisting of primary amines, secondary amines, and quaternary-ammonium bases, until substantially all the iatty acids of the glyceride have combined therewith and then separating the vitamin in concentrated form from the reaction mixture.

2. The process 01' preparing a fat-soluble vitamin concentrate which comprises reacting a glyceride fat which contains a fat-soluble vitamin with at least approximately stoichiometric amounts of a primary amine, until substantially all the fatty acids of the glyceride have combined therewith and then separating the vitamin from the reaction mixture.

3. The process or preparing a fat-soluble vitamin concentrate which comprises reacting a glyceride fat which contains a fat-soluble vita min with approximately stoichiometric amounts or a member of the group consisting of primary amines, secondary amines, and quaternary amamounts of a primary amine at a temperature of between about 80 and 150, until substantially all the fatty acids of the glyceride have combined therewith, and then separating the vitamin from the reaction mixture.

5. The proces of preparing vitamin A concentrate which comprises reacting a glyceride fat derived from a fish which contains vitamin A with at least approximately stoichiometric amounts of a member of the group consisting of primary amines, secondary amines, and quaternary'ammonium bases, until substantially all the fatty acids of the glyceride have combined therewith and then separating the vitamin A in concentrated form from the reaction mixture.

. 6. The process of preparing vitamin A concen. trate which comprises reacting a glyceride fat derived from a flshwhich contains vitamin A with at least approximately stoichiometric amounts of a member or the group consisting of primary amines, secondary amines, and quaternary ammonium bases, until substantially all the fatty acids or the glyceride have combined therewith and then subjecting the reaction mixture to highvacuum unobstructed path distillation and separating a vitamin A concentrate as a distillate.

'7. The process of preparing a fat-soluble vitamin concentrate which comprises reacting a glyceride tat which contains a fat-soluble vitamin with 1 to 10 molecular equivalents of a member oi the group consisting of primary amines, secondary amines, and quaternary ammomum bases, until substantially all the fatty acids of the glyceride have combined therewith and separating the vitamin from the reaction product. 8. The process of preparing a fat-soluble vitamin concentrate which comprises reacting a glyceride tat which contains a fat-soluble vitamin with at least approximately stoichiometric amounts of. a member of the group consisting oi primary amines, secondary amines, and quaternary ammonium bases until substantially all the fatty acids of the glyceride have combined therewith at a temperature of about for a period of time between about 2 to 20 hours, and then separating the vitamin from the reaction product.

9. The process of preparing a fat-soluble vitamin concentrate which comprises reacting a glyceride fat derived from a fish and containing a fat-soluble vitamin with l to 10 molecular equivalents of a primary amine until substantially all the fatty acids of the glyceride have combined therewith at a temperature of about 100 for a period of time between about 2 to 20 hours, and

separating a vitamin in concentrated form from EDGAR M. SHANTZ. 

